The molecular mechanisms which govern immune tolerance to self tissues are still not understood; the breakdown of these mechanisms can lead to autoimmune diseases including insulin-dependent diabetes. CD8+ cytotoxic T (Tc) cells play a key role in pathogenesis of autoimmune diabetes in mouse and human. Why Tc cells are normally tolerant to pancreatic antigens and how autoreactive Tc cells become activated in autoimmune diabetes is unclear. Cross-priming has been indicated as a common mechanism for MHC class I presentation of cell-associated antigen. It has been shown by use of RIP-Ova mice that chicken ovalbumin protein expressed in the pancreatic islets can be presented by dendritic cells in draining lymph nodes to Tc cells, which results in cross-tolerance of self-reactive CD8 cells. The decision of T cell activation vs. tolerance is most importantly regulated by costimulatory signals on antigen-presenting cells (APC). In the current study, we propose to test our hypothesis that Tc cell activation and tolerance are tightly regulated by the positive and negative costimulatory molecules by use of the RIP-ova mice. First, we will analyze the roles of CD28 and ICOS in regulation of Tc cell activation. We will transfer ICOS+/+ and ICOS-/- OT-I cells into RIP-mOva transgenic mice sufficient or deficient in the B7.1 and B7.2 genes, and their activation and tolerance will be examined. Secondly, we will examine the roles of PDL2, B7-H3 and B7S1 in regulation of Tc cell tolerance. We will transfer OT-I T cells into RIP-mOva mice with or without treatment with anti-PDL2, BT-H3 or B7S1 blocking antibodies and the activation or tolerance of the transferred cells wilt be examined. This study will advance our understanding on costimulatory regulation of Tc activation and tolerance, and may likely to suggest means to modulate immune tolerance to tissue antigens. [unreadable] [unreadable]